Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
Company Limited by Guarantee l Registered in England No. 1039582 l Registered Office Charles Darwin House, 12 Roger Street, WC1N 2JU, UK
Registered as a Charity: 264017 (England & Wales); SC039250 (Scotland)
16737403-file00.docx
Validation of a single-step, single-tube reverse transcription-loop-mediated 1
isothermal amplification assay for rapid detection of SARS-CoV-2 RNA 2
3
1.1 Author names 4
Jean Y. H. Lee1,2, Nickala Best3, Julie McAuley1, Jessica L. Porter1, Torsten Seemann1,4, Mark B. 5
Schultz4, Michelle Sait4, Nicole Orlando4, Karolina Mercoulia4, Susan A. Ballard4, Julian Druce5, 6
Thomas Tran5, Mike G. Catton5, Melinda J. Pryor6, Huanhuan L. Cui6, Angela Luttick6, Sean 7
McDonald3, Arran Greenhalgh3, Jason C. Kwong1,7, Norelle L. Sherry4,7, Maryza Graham8, Tuyet 8
Hoang1,4, Marion Herisse1, Sacha J. Pidot1, Deborah A. Williamson1,4,9, Benjamin P. Howden1,4,7, Ian 9
R. Monk1,#, and Timothy P. Stinear1,*,# 10
11
1.2 Affiliation 12
1. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty 13
Institute for Infection and Immunity, Melbourne, Victoria, Australia. 14
2. Department of Infectious Diseases, Monash Health, Clayton, Victoria, Australia. 15
3. GeneWorks Pty Ltd, Thebarton, South Australia, Australia. 16
4. Microbiological Diagnostic Unit Public Health Laboratory, University of Melbourne at the Peter 17
Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia. 18
5. Victorian Infectious Diseases Reference Laboratory, Melbourne Health at the Peter Doherty 19
Institute for Infection and Immunity, Melbourne, Victoria, Australia. 20
6. 360Biolabs, Melbourne, Victoria, Australia 21
7. Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia 22
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 2 of 55
16737403-file00.docx
8. Department of Microbiology, Monash Health, Clayton, Victoria, Australia 23
9. Melbourne Health, Melbourne, Victoria, Australia 24
25
1.3 Corresponding author 26
* Corresponding author, [email protected] 27
# Joint senior authors 28
29
1.4 Keyword 30
SARS-CoV-2, RT-LAMP, universal transport media, nasopharyngeal swabs 31
32
2. Abstract 33
Introduction: The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for 34
RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has 35
necessitated the exploration of alternative diagnostic options to alleviate supply chain issues. 36
Aim: To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- 37
LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs. 38
Methodology: We used a commercial RT-LAMP mastermix from OptiGene Ltd in combination with 39
a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A 40
single-tube, single-step fluorescence assay was implemented whereby as little as 1 μL of universal 41
transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing 42
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 3 of 55
16737403-file00.docx
the requirement for RNA purification. Amplification and detection could be conducted in any 43
thermocycler capable of holding 65°C for 30 minutes and measure fluorescence in the FAM channel 44
at one-minute intervals. 45
Results: Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene 46
RT-qPCR revealed assay sensitivity and specificity of 87% and 100%, respectively. Results were fast, 47
with an average time-to-positive (Tp) for 93 clinical samples of 14 minutes (SD +7 minutes). Using 48
dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA 49
guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 50
Tissue Culture Infectious Dose 50 per ml (TCID50 mL-1), with satisfactory assay sensitivity and 51
specificity. A comparison of 20 clinical specimens between four laboratories showed excellent 52
interlaboratory concordance; performing equally well on three different, commonly used 53
thermocyclers, pointing to the robustness of the assay. 54
Conclusion: With a simplified workflow, N1-STOP-LAMP is a powerful, scalable option for specific 55
and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium 56
against COVID-19. 57
58
3. Data summary 59
The authors confirm all supporting data, code and protocols have been provided within the article or 60
through supplementary data files. 61
62
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 4 of 55
16737403-file00.docx
4. Introduction 63
The current SARS-CoV-2 pandemic has created an unprecedented global demand for rapid diagnostic 64
testing. Most countries are employing real-time quantitative PCR (RT-qPCR) for confirmation of 65
infection [1-7]. Consequently, a global shortage of RNA extraction kits as well as RT-qPCR assay kits 66
and their associated reagents has ensued [8-10]. Therefore, alternative diagnostics not dependent on 67
these commonly used materials are required. First described 20 years ago by Notomi et al., the loop-68
mediated isothermal amplification (LAMP) assay is robust, rapid and straightforward, yet retains high 69
sensitivity and specificity [11]. These features have seen the LAMP assay and the inclusion of a 70
reverse transcriptase (RT-LAMP) implemented for a broad range of molecular diagnostic applications 71
extending from infectious diseases, including detection of the original SARS-CoV-1 virus [12], 72
bacteria and parasites [13-15] to cancer [16]. The advantages of RT-LAMP include; using different 73
reagents than RT-qPCR, the potential for direct processing of samples without the need for prior RNA 74
extraction and an extremely rapid turn-around time. Several groups have now described different RT-75
LAMP assays for detection of SARS-CoV-2 RNA [17-24]. 76
77
In this study, we developed a RT-LAMP assay that targets the CDC N1 region of the SARS-CoV-2 78
nucleocapsid gene (N-gene) [3] and used a commercial mastermix from OptiGene Ltd. This mix 79
contains a proprietary reverse transcriptase for cDNA synthesis and the thermophilic GspSSD strand-80
displacing polymerase/reverse transcriptase for DNA amplification (www.optigene.co.uk) with a 81
dsDNA intercalating fluorescent dye. Detection was achieved by measuring the increase in 82
fluorescence as amplification products accumulate. The N1 gene Single Tube Optigene LAMP assay 83
(hereafter called N1-STOP-LAMP) was assessed for the direct detection of SARS-CoV-2 RNA, 84
following FDA Policy for Diagnostic Tests for Coronavirus Disease-2019 during the Public Health 85
Emergency against the four parameters and acceptance criteria summarised in Table 1 [5]. Validation 86
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 5 of 55
16737403-file00.docx
samples were human upper respiratory tract specimens, collected using nasopharyngeal flocked swabs 87
stored in universal transport media (UTM). 88
89
5. Methods 90
Specimen collection and handling 91
Nasopharyngeal swabs were collected by qualified healthcare professionals from patients meeting the 92
epidemiological and clinical criteria as specified by the Victorian Department of Health and Human 93
Services at the time of swab collection [1] between the 23rd of March 2020 and 4th of April 2020. 94
Copan flocked swabs collected and directly inoculated on site in either 1 mL or 3 mL of UTM® 95
(Catalogue Nos., 330C and 350C respectively) were used. Samples were collected at metropolitan 96
hospitals in Melbourne, Victoria, Australia, and transported to the Doherty Institute Public Health 97
Laboratories for further testing as per World Health Organization recommendations [6]. All swabs 98
were processed in a class II biological safety cabinet. 99
100
Cell culture and SARS-CoV-2 RNA extraction 101
Vero cells (within 30 passages from the original American Type Culture Collection [ATCC] stock) 102
were maintained in Minimal Essential Media (MEM) supplemented with 10% heat-inactivated foetal 103
bovine serum (FBS), 10 µM HEPES, 2 mM glutamine and antibiotics. Cell cultures were maintained 104
at 37oC in a 5% CO2 incubator. All virus infection cultures were conducted within the High 105
Containment Facilities in a PC3 laboratory at the Doherty Institute. To generate stocks of SARS-CoV-106
2, confluent Vero cell monolayers were washed once with MEM without FBS (infection media) then 107
infected with a known amount SARS-CoV-2 virus originally isolated from a patient [25]. After 1h 108
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 6 of 55
16737403-file00.docx
incubation at 37oC in a 5% CO2 incubator to enable virus binding, infection media containing 1 mg 109
mL-1 TPCK-trypsin was added, and flasks returned to the incubator. After 3d incubation and 110
microscopic confirmation of widespread cytopathic effect (CPE), the supernatants were harvested, 111
and filter sterilised through a 0.45 µm syringe filter. To assess infectious SARS-CoV-2 viral titres, 112
both Tissue Culture Infectious Dose 50 (TCID50) and plaque assays were performed. Briefly, serial 113
dilutions of the stock virus were added to washed monolayers of Vero cells. After 1h incubation to 114
allow virus to adhere, for the TCID50 assay, infection media containing 1 µg mL-1 TPCK-Trypsin was 115
added, while for the plaque assay the infected cell monolayer was overlaid with Leibovitz-15 (L15) 116
media supplemented with 0.9% agarose (DNA grade, Sigma), antibiotics and 2 µg mL-1 TPCK 117
Trypsin. After 3d incubation, the dilution of stock required to cause CPE in at least 50% of wells 118
(TCID50) was determined via back calculation of microscopic confirmation of CPE in wells for a 119
given dilution. For quantitation via plaque assay, plaques present in the monolayer were 120
macroscopically visualised, individually counted and back calculated for each given dilution to 121
determine the number of Plaque Forming Units (PFU) per mL in the original stock. Stocks of SARS-122
CoV-2 used by this study had a TCID50 of 5.4x105 mL-1 and plaque assay gave 9.78x105 PFU mL-1. 123
To heat inactivate the virus, 200 µL of neat stock was heated to 60oC for 30 min, then cooled. 124
Inactivation was confirmed via complete lack of CPE and plaque formation using both TCID50 and 125
plaque assays. To prepare SARS-CoV-2 RNA from stocks, 500 µL aliquots were thawed and RNA 126
extracted using the RNeasy mini Kit (Qiagen) according to the manufacturer’s specifications, with an 127
elution volume of 50 µL. Based on RNA concentrations, the total virus harvested from Vero cell 128
culture was 3.1x109 copies mL-1, suggesting a high number of non-infectious virus particles in the 129
virus stocks. 130
131
RNA extraction from UTM for RT-qPCR 132
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 7 of 55
16737403-file00.docx
A 200 µL aliquot of Copan UTM® was processed through the QIAsymphony DSP Virus/Pathogen 133
Mini Kit (Qiagen, Cat# 937036) following manufacturer’s instructions on the QIAsymphony SP 134
instrument. The RNA was eluted in 60 µL of recommended buffer. 135
136
RNA extraction from UTM SPRI beads for N1-STOP-LAMP 137
Solid-phase reversible immobilisation (SPRI) on carboxylated paramagnetic beads (Sera-Mag™ 138
Magnetic SpeedBeads™, from GE Healthcare) were prepared for RNA binding as described 139
(https://openwetware.org/wiki/SPRI_bead_mix). RNA purification was performed in 96-well plates 140
with initial lysis by the addition of 25 µL of 6GTD lysis buffer (7.08 g 10 mL-1 guanidine thiocyanate 141
(6M), made up to 8.2 mL with water, 1 mL Tris HCL [pH 8.0] and 800 µL of 1M dithiothreitol), 142
mixed by pipetting ten times and incubation at room temperature for 1 min [26]. To this, 75 µL of 143
100% ethyl alcohol and 20 µL of prepared SPRI beads were added, mixed by pipetting ten times and 144
incubated at room temperature for 5 min. The RNA-bead complex was then immobilised by placing 145
the 96-well plate on a magnetic rack and incubated again at room temperature for 5 min. The 146
supernatant was then discarded, and the beads washed twice in 200 µL of freshly prepared 80% ethyl 147
alcohol (v/v) with 30 sec room temperature incubation between each wash. The beads were air-dried 148
for 2 min at room temperature before RNA was eluted by the addition of 20 µL of nuclease-free 149
water. 150
151
E-gene RT-qPCR 152
A one-step RT-qPCR was conducted with the primers as described by Corman et al., targeting the 153
viral envelope E-gene of SARS-CoV-2 E_Sarbeco_F: 5’-154
ACAGGTACGTTAATAGTTAATAGCGT-3’, 5’-E_Sarbeco_R: 155
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 8 of 55
16737403-file00.docx
ATATTGCAGCAGTACGCACACA-3’, E_Sarbeco_P: 5’-FAM-156
ACACTAGCCATCCTTACTGCGCTTCG-BHQ1-3’) [27]. A 20 µL reaction was assembled 157
consisting of 1x qScript XLT One-Step RT-qPCR ToughMix Low ROX (2x) (QuantaBio), 400 nM 158
E_Sarbeco_F, 400 nM E_Sarbeco_R 200 nM E_Sarbeco_P (Probe) and 5 µL of purified RNA. The 159
following program was conducted on an ABI 7500 Fast instrument: 55°C for 10 min for reverse 160
transcription, 1 cycle of 95°C for 3 min and then 45 cycles of 95°C for 15 sec and 58°C for 30 sec. 161
162
N1-STOP-LAMP 163
A 50-reaction bottle of dried Reverse Transcriptase Isothermal Mastermix (Optigene, ISO-DR004-164
RT) was rehydrated with 750 μL of resuspension buffer and vortexed gently to mix. The mix contains 165
both a proprietary reverse transcriptase and the GspSSD LF DNA polymerase that has both reverse 166
transcriptase and strand-displacing DNA polymerase activity. The N1-STOP-LAMP assay uses six 167
standard LAMP oligonucleotide primers that target the sequence spanning the CDC N1 region of the 168
SARS-CoV-2 nucleocapsid gene. Sequences of the primers are available upon request (GeneWorks 169
Ltd). Each N1-STOP-LAMP reaction contained: 15 μL of mastermix, 5 μL of 5x primer stock and 4 170
μL of water. Mastermix was reconstituted in a separate biological safety cabinet to that used for 171
template addition. The source of RNA template for the reaction consisted of either 1 μL of purified 172
SARS-CoV-2 RNA, 1 μL of SARS-CoV-2 purified virus or 1 μL of UTM from a nasopharyngeal 173
swab. A no-template control (1 μL water) was included in all runs. Reactions were assembled in 174
either 8-tube Genie strips (OP-00008, OptiGene Ltd) or 96-MicroAmp-Fast-Optical reaction plate 175
(Applied Biosystems). Strip tubes reactions were capped, or for 96-MicroAmp-Fast-Optical reaction 176
plates, sealed with MicroAmp Optical adhesive film (Applied Biosystems) and tapped to remove 177
bubbles. Strip tubes were loaded onto the Genie-II or Genie-III (OptiGene Ltd) and 96-well plates run 178
on a QuantStudio 7 thermocycler (ThermoFisher). Reactions were incubated at 65°C for 30 min with 179
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 9 of 55
16737403-file00.docx
fluorescence acquisition every 30 sec (Genie instruments) or 1 min (QuantStudio 7). A positive result 180
was indicated by an increase in fluorescence at an emission wavelength of 540 nm (FAM channel) 181
above a defined threshold, recorded as time-to-positive (Tp) expressed in min:sec. 182
183
Dilution series of purified SARS-CoV-2 184
A virus stock with of 5.4 x105 TCID50 mL-1 was serially diluted to 10-6 in a generic UTM (comprising 185
per liter: 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4, 4 mL 0.5% phenol red 0.5%, 5 g 186
gelatin, 950 mL tissue culture water, 1 mL Fungizone [5 mg mL-1], 20 mL penicillin/streptomycin) in 187
biological triplicates, with the 10-1 through to the 10-6 dilutions tested by N1-STOP-LAMP (1 μL). 188
RNA was extracted from a 200 μL aliquot of each dilution as described above and 5 μL of the 189
purified RNA used as template in E-gene RT-qPCR or N1-STOP-LAMP assay. 190
191
Interlaboratory comparison of N1-STOP-LAMP 192
A panel of 20 blinded clinical samples (13 positive and 7 negative) with cycle threshold (Ct) values 193
previously established by E-gene RT-qPCR were aliquoted and distributed to three different 194
laboratories for independent testing by N1-STOP-LAMP assay (Table S2). 195
196
Biological specificity 197
To test for cross reactivity of the N1-STOP-LAMP assay, a control panel of respiratory pathogens 198
(NATRPC2-BIO, ZeptoMetrix) was screened (Table S3). A 1 μL aliquot of NATrol RP1 or RP2, or 199
samples spiked with SARS-CoV-2 virus were used as template for N1-STOP-LAMP. 200
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 10 of 55
16737403-file00.docx
201
In silico nucleotide sequence comparisons of the N1 region of SARS-CoV-2 202
To assess the inclusivity and exclusivity of the N1 region targeted by the LAMP assay 2755 publicly 203
available SARS-CoV-2 genomes were downloaded and filtered to remove entries of less than 29 kb 204
using Seqtk seq (v1.3-r106). Sequence gaps were replaced with ‘N’ using Seqkit (v0.12.0). After this 205
quality control step, homology searches were then conducted using NCBI BLAST+ blastn using 206
Genepuller.pl (https://github.com/tseemann/bioinfo-scripts/blob/master/bin/gene-puller.pl) to find the 207
region in each of the remaining 2738 genomes matching the 5’ region of the N1 sequence. The 208
resulting sequence coordinates of those hits were then used to extract the 240 bp region from all 2738 209
genomes using Genepuller.pl and aligned with Clustalo (v1.2.4). The alignment was visualised with 210
Mesquite (v3.61). 211
212
Statistical analysis 213
Data analysis was managed using GraphPad Prism (v8.4.1). Sensitivity and specificity testing were 214
performed using the Wilson/Brown hybrid method as deployed in GraphPad Prism. 215
216
6. Results 217
Assessing detection sensitivity of N1-STOP-LAMP using purified RNA. 218
We began by assessing the limit of detection (LoD) of N1-STOP-LAMP under optimum conditions 219
using a 10-fold dilution series of purified RNA, prepared from a titred SARS-CoV-2 virus stock. 220
Although LAMP assays are not strictly quantitative, a positive correlation between Tp and RNA 221
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 11 of 55
16737403-file00.docx
concentration was evident. This was indicated by an absolute detection threshold between 50 and 500 222
viral genome copies per reaction. We achieved a reliable detection of 5/5 replicates at 500 viral 223
genome copies and detection of 4/5 replicates at 50 viral genome copies per reaction (Fig. 1A). Of 224
note, this threshold is likely an underestimate of the true detection limit, as we assumed all RNA 225
yielded from the viral stock generated from the Vero cell supernatant was of viral origin. Using 226
TCID50, the absolute LoD of N1-STOP-LAMP was between 0.001 and 0.01 TCID50 per reaction 227
(equivalent to 1-10 TCID50 mL-1). 228
229
Direct detection of SARS-CoV-2 RNA in clinical specimens and specimen inactivation 230
The nasopharyngeal specimens received by our public health laboratories were collected using Copan 231
flocked swabs in either 1 mL or 3 mL of UTM®. To assess the impact of sample matrix on the LAMP 232
assay, we performed pilot experiments with UTM from swabs taken from SARS-CoV-2 negative 233
specimens, adding increasing amounts of UTM to N1-STOP-LAMP. Sterile UTM had no impact on 234
N1-STOP-LAMP (data not shown). However, the nasopharyngeal secretions in patient samples were 235
observed to be inhibitory. Most pronounced when 5 µL of patient sample was used as the direct RNA 236
template (Fig. 1B), but relieved at a 1/5 dilution of the patient sample, thus 5 µL of a 1/5 dilution of 237
the patient sample or a 1 µL of neat patient sample was selected as the optimum template volume for 238
N1-STOP-LAMP (Fig. 2A). Experiments were also conducted using dry swabs eluted in PBS. We 239
tested elution in 0.5 mL, 1.0 mL and 1.5 mL and found that an elution volume of 1.5 mL of PBS was 240
a good compromise between unnecessary dilution of potential virus in the sample and sufficient 241
dilution to alleviate assay inhibition (data not shown). To reduce the risk associated with handling 242
clinical specimens containing infectious SARS-CoV-2, we also assessed the impact of a heat 243
inactivation step on detection sensitivity. Pre-treatment at 60°C for 30 min led to a >5-log10 244
inactivation of the virus accessed by PFU and TCID50 determination (data not shown). No impact on 245
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 12 of 55
16737403-file00.docx
N1-STOP-LAMP detection sensitivity was observed, with equivalent Tp between each treatment 246
(Table 2, Fig. 1C). 247
248
Comparative detection sensitivity of N1-STOP-LAMP versus E-gene RT-qPCR. 249
The N1-STOP-LAMP assay was then directly compared with E-gene RT-qPCR, using a dilution of 250
titred SARS-CoV-2 virus stock in UTM across a 6-log10 dilution series. A RT-qPCR calibration curve 251
was established from triplicate extraction experiments for each of the six dilutions (Fig. 2A). The 252
assays were prepared with 5 µL of purified viral RNA as template for both the N1-STOP-LAMP and 253
RT-qPCR assays. A 1 µL and 5 µL aliquot of each virus dilution in UTM was also tested directly in 254
the N1-STOP-LAMP assay (i.e. without RNA extraction). The side-by-side comparisons showed E-255
gene RT-qPCR was up to 1-log10 more sensitive than N1-STOP-LAMP, with RT-qPCR detecting 2/3 256
replicates at the lowest dilution of 0.54 TCID50 mL-1 and N1-STOP-LAMP detecting no viral RNA at 257
this concentration (Table 3, Fig. 2). At 54 TCID50 mL-1, the RT-qPCR and N1-STOP-LAMP detected 258
3/3 replicates. There was no difference in N1-STOP-LAMP sensitivity using either 5 µL UTM added 259
directly to the test or purified RNA, but the assay lost another 1-log10 sensitivity where 1 µL of neat 260
UTM was added directly to the N1-STOP-LAMP reaction (Fig. 2B). 261
262
Establishing N1-STOP-LAMP limit of detection (LoD) 263
The FDA guidelines for implementation of emergency-use diagnostics defines LoD as the lowest 264
concentration at which 19/20 replicates are positive (Table 1). Informed by the previous experiment 265
(Fig. 2B), we selected a SARS-CoV-2 concentration of 54 TCID50 mL-1 (in UTM) and tested 20x 1 µL 266
aliquots by N1-STOP-LAMP. We observed 20/20 positive reactions with an average Tp of 15.8 mins, 267
inter-quartile range 12-19 mins and coefficient of variation of 31.18% (Fig. 3A). 268
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 13 of 55
16737403-file00.docx
269
Evaluation of N1-STOP-LAMP against FDA criteria. 270
The FDA guidelines for implementation of emergency-use diagnostics also require an assessment of 271
assay performance using at least 30 contrived positive and 30 negative clinical specimens (authentic 272
or contrived), with at least 20 of the positives at a concentration of 1-2x the LoD (Table 1). We 273
obtained 30 nasopharyngeal dry swabs from healthy, anonymous volunteers. Swabs were eluted in 1.5 274
mL of PBS and 1 µL aliquots were tested directly by N1-STOP-LAMP. All eluates were negative. We 275
then spiked the eluates with SARS-CoV-2 virus at 1x and 2x the LoD (approx. 50 and 100 TCID50 276
mL-1, respectively). Results showed that N1-STOP-LAMP detected 20/20 samples spiked with 1x the 277
assay LoD and 10/10 samples at 2x LoD (Fig. 3B, Table 4). N1-STOP-LAMP thus meets the FDA 278
Clinical Evaluation criteria (Table 1). 279
280
Clinical specimen evaluation of N1-STOP-LAMP against E-gene RT-qPCR gold standard 281
We then sought to evaluate N1-STOP-LAMP performance against E-gene RT-qPCR, setting the latter 282
assay as the current ‘gold standard’. We directly screened 50 negative clinical specimens and 107 283
positive nasopharyngeal clinical specimens as established by E-gene RT-qPCR (Table S1). Results 284
showed N1-STOP-LAMP assay sensitivity of 87%, specificity of 100%, positive predictive value of 285
100% and negative predictive value of 78% (Table 5, Fig. 3). As noted during initial sensitivity 286
experiments (Table 3), a positive correlation was observed between N1-STOP-LAMP Tp and E-gene 287
RT-qPCR Ct (Fig. 4). The average Tp among the 93 N1-LAMP-STOP positive specimens was 14 min 288
(SD + 7 min). For the false-negative samples with sufficient clinical material remaining (5 out of 14), 289
RNA extraction using a simple, rapid magnetic bead purification protocol (takes 20 min) was 290
performed, then N1-STOP-LAMP repeated. Four of the 5 samples returned a positive result with Tp 291
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 14 of 55
16737403-file00.docx
range from 10:00 – 25:30 min:sec (Table S1). Indicating that the reduced sensitivity of N1-STOP-292
LAMP compared to RT-qPCR can be improved upon by using samples with a higher RNA template 293
concentration and purity with only a small increase in time to results. 294
295
Confirmation of N1-STOP-LAMP inclusivity and exclusivity 296
The FDA guidelines for implementation of emergency-use diagnostics require an assessment of 297
inclusivity and exclusivity of the oligonucleotide primers used in an assay. To assess the former, we 298
performed in silico screening of the region spanned by the six primers used for the N1-STOP-LAMP 299
assay against all available SARS-CoV-2 genome sequences (Supplementary data file 1). This 240 bp 300
sequence spans the CDC N1 region of the SARS-CoV-2 N-gene [3]. Alignment of this region against 301
2738 genomes revealed 100% conservation amongst all entries, showing that this a conserved target 302
sequence and that the primers used will perform equally across all identified lineages of the virus. 303
304
To assess the exclusivity criterion, we used a nucleotide BLAST search of the N1 region against the 305
NCBI Genbank nt database and observed no non-SARS-CoV-2 sequence matches above 80% 306
nucleotide identity, in-line with FDA cross-reactivity requirement. Further to this analysis, we also 307
performed in vitro testing with the NATRPC2-BIO (ZeptoMetrix) specificity panel, a control panel 308
consisting of 22 common respiratory pathogens. None of these pathogens were detected by N1-STOP-309
LAMP (Table S3). Thus, both in silico and in vitro testing confirmed the specificity of N1-STOP-310
LAMP for SARS-CoV-2. 311
312
Interlaboratory comparisons 313
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 15 of 55
16737403-file00.docx
In order to explore the ability of other laboratories to readily deploy N1-STOP-LAMP, we prepared 314
an external-quality-assessment (EQA) panel of 20 clinical specimens (Table S2). These were previous 315
positive and negative clinical samples as assessed by E-gene RT-qPCR that had been heat-inactivated 316
for safety. These specimens covered a range of virus titres (Ct values 16.3 – 30.6). The panel was 317
tested simultaneously in four different laboratories (Labs A – D). The results showed excellent 318
correspondence between all laboratories for ten positive samples, the majority with E-gene RT-qPCR 319
Ct values <26 (Fig. 5, Table S2). Above this Ct (i.e. those samples with lower virus titres), the 320
laboratories returned variably positive results (Samples 14, 16 & 18), reflecting that these specimens 321
had virus concentrations at or beyond the N1-STOP-LAMP LoD (Table S2). Concordance among the 322
seven negative results was overall very good, however one laboratory (Lab C) returned a false 323
positive result (Sample 2), highlighting the issue of contamination with sensitive molecular tests 324
(Table S2). Of note, the laboratories used a range of different instruments for amplification/detection 325
including: OptiGene Genie II and III, BioRad CFX and ThermoFisher Quantstudio 7. This trial 326
suggests that N1-STOP-LAMP is a robust and transferrable assay format. 327
328
7. Discussion 329
A critical component of an effective COVID-19 pandemic response is the rapid and robust detection 330
of positive clinical samples [28]. This has required massively upscaled SARS-CoV-2 diagnostic 331
capabilities. Particularly, a worldwide focus on developing improved technologies [29]. There have 332
been more than 20 molecular tests recently receiving FDA Emergency Use Authorisations (EUAs) 333
[5]. In this current study, we demonstrate the potential of N1-STOP-LAMP. We focused efforts on the 334
LAMP assay because it has been previously employed for the rapid and robust detection of numerous 335
RNA viruses including Zika, Chikungunya, Influenza and SARS-CoV-1 [12, 30-33] and was of great 336
assistance during these outbreaks, particularly in resource constrained settings. Advantages of LAMP 337
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 16 of 55
16737403-file00.docx
testing include rapid turnaround time, ease of implementation, non-standard reagent use and potential 338
utility at point of care [19-21]. To date, there has been limited detail on the performance 339
characteristics of emerging RT-LAMP based tests in head-to-head comparisons with gold standard 340
assays. Such information is critical to ensuring the safe deployment of new tests, given the clinical 341
and public health consequences of erroneous test results. 342
343
Here we have shown the specific detection of SARS-CoV-2 RNA directly from clinical swabs 344
without the need for RNA purification, using a LAMP assay targeting the N1 region of the SARS-345
CoV-2 N-gene in conjunction with the reverse transcriptase/GspSSD LF DNA polymerase mastermix 346
from OptiGene Ltd. With a detection limit of 54 TCID50 mL-1 the N1-STOP-LAMP assay met each of 347
the four FDA criteria for EUA. Based on reported data, as opposed to head-to-head comparisons, the 348
N1-STOP-LAMP detection limit, is higher than several recently reported FDA-EUA COVID-19 tests. 349
Expressed as virus copies per mL the N1-STOP-LAMP LoD is estimated at 54,000 virus copies per 350
mL (assumes our TCID50 underestimates virus copy number by 1000-fold, see methods), compared to 351
Cepheid GeneXpert Xpress 250 virus copies per mL or Abbott ID NOW COVID-19 test 125 virus 352
copies per mL. Other EUA assays reporting LoDs similar to N1-STOP-LAMP include the Luminex 353
ARIES SARS-CoV-2 assay (75,000 copies per mL) and the GenMark ePlex SARS-CoV-2 test 354
(100,000 copies per mL). Given these performance metrics, and the test’s high positive predictive 355
value (100%), we see N1-STOP-LAMP as well suited for widespread screening of large populations 356
when COVID-19 prevalence is low. N1-STOP-LAMP could be used in large-scale, national testing 357
programs to support aggressive COVID-19 contact tracing and disease suppression or elimination 358
activities. The test could also play a role in near-point-of-care settings, such as outbreak investigation 359
in hospitals and nursing homes, providing rapid-turnaround-time of results for health-care workers 360
and highly vulnerable patients. The relatively simple assay format of N1-STOP-LAMP is also suitable 361
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 17 of 55
16737403-file00.docx
for deployment in lower and middle-income countries, where access to sophisticated laboratory 362
infrastructure is limited. 363
364
During this evaluation we noted opportunities for improvement of N1-STOP-LAMP. We observed 365
that although 1 µL of UTM was tolerated in the OptiGene RT-LAMP formulation, larger quantities of 366
sample matrix (UTM or PBS containing additional human material from the nasopharyngeal swab) 367
were inhibitory to the RT-LAMP reaction. If more than 1 µL of spiked (purified virus or RNA as 368
template) sample matrix was used in the assay, no amplification was observed (Fig. 1B). However, 369
under ideal conditions (i.e., no sample inhibition from human components), the impact of using an 370
increased template volume was shown, where 5 µL of UTM added directly to the N1-STOP-LAMP 371
reaction was equal to the sensitivity of using purified RNA (Fig. 2B). Furthermore, addition of a 372
simple RNA purification step, that does not require commercial kits, improved the detection 373
sensitivity of N1-STOP-LAMP to rival RT-qPCR. Our follow-up of five false-negative results (Table 374
5, Fig. 3C), demonstrated the feasibility of including a simple, scalable and rapid magnetic-bead RNA 375
purification step [26], with which we were able to detect SARS-CoV-2 RNA with N1-STOP-LAMP 376
in 4/5 UTM specimens that had previously tested negative by the assay (Table S1). Other 377
opportunities for improvement include the use of specific fluorescent probes to detect amplification 378
products, thus facilitating multiplex reactions and the inclusion of an internal amplification control 379
within each test [24,34]. 380
381
As mentioned, recognised strengths of the LAMP assay include the rapid time to test result, with the 382
majority of the positive clinical samples yielding a result in under 15 min, and the robust test format, 383
testing directly from the swab eluate. Our preferred specimen type for N1-STOP-LAMP is a dry 384
nasopharyngeal swab. Detailed examination of optimum swabs for the detection of influenza and 385
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 18 of 55
16737403-file00.docx
other RNA viruses has shown dry swabs offer superior performance [35]. In the current study we have 386
shown that N1-STOP-LAMP has satisfactory performance using dry swabs eluted in 1.5 mL of PBS 387
(Fig. 3B); a format that potentially doubles the effective concentration of virus in the sample 388
compared to 3 mL of UTM in the Copan format. Another strength of N1-STOP-LAMP is that the 389
assay readily scales from small sample numbers (e.g. 8-well with portable detection unit) to high 390
throughput (run with sample robotics and 96-well thermocyclers in a centralised laboratory). 391
392
While establishing the assay, we noted susceptibility to contamination that was easily addressed by 393
the standard precautions used to prevent template carryover for any nucleic acid amplification test, 394
such as: physical separation of mastermix preparation from sample inoculation; not opening post-395
amplification reaction tubes or plates; and inclusion of negative extraction controls. Addition of 396
uracil-N-glycosylase has been reported as a strategy to limit the risk of amplicon contamination, 397
however this is associated with a loss of detection sensitivity [36]. 398
399
In this report we have shown N1-STOP-LAMP is robust diagnostic test for the specific and rapid 400
detection of SARS-CoV-2. It is an alternative molecular test for SARS-CoV-2 that can be readily and 401
seamlessly deployed, particularly when access to standard RT-qPCR based approaches are limited. 402
403
8. Author statements 404
8.1 Authors and contributors 405
Jean Lee: 406
Conceptualisation 407
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 19 of 55
16737403-file00.docx
Data curation 408
Formal analysis 409
Funding acquisition 410
Investigation 411
Methodology 412
Project administration 413
Resources 414
Software 415
Supervision 416
Validation 417
Visualisation 418
Writing - original draft 419
Writing - review & editing 420
421
Nickala Best: 422
Conceptualisation 423
Data curation 424
Formal analysis 425
Investigation 426
Methodology 427
Resources 428
Validation 429
Visualisation 430
Writing - original draft 431
Writing - review & editing 432
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 20 of 55
16737403-file00.docx
433
Julie McAuley: 434
Conceptualisation 435
Data curation 436
Formal analysis 437
Investigation 438
Methodology 439
Resources 440
Validation 441
Writing - review & editing 442
443
Jessica Porter: 444
Data curation 445
Formal analysis 446
Investigation 447
Methodology 448
Writing - review & editing 449
450
Torsten Seemann: 451
Conceptualisation 452
Investigation 453
Methodology 454
Software 455
456
Mark Schultz: 457
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 21 of 55
16737403-file00.docx
Conceptualisation 458
Investigation 459
Methodology 460
Software 461
462
Michelle Sait: 463
Formal analysis 464
Investigation 465
Methodology 466
Software 467
468
Nicole Orlando: 469
Formal analysis 470
Investigation 471
Methodology 472
473
Karolina Mercoulia: 474
Data curation 475
Investigation 476
Methodology 477
478
Susan Ballard: 479
Conceptualisation 480
Data curation 481
Formal analysis 482
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 22 of 55
16737403-file00.docx
Investigation 483
Methodology 484
485
Julian Druce: 486
Conceptualisation 487
Data curation 488
Formal analysis 489
Investigation 490
Methodology 491
492
Thomas Tran: 493
Investigation 494
Methodology 495
496
Mike Catton: 497
Conceptualisation 498
Investigation 499
Supervision 500
Writing - review & editing 501
502
Melinda Pryor: 503
Conceptualisation 504
Investigation 505
Methodology 506
Supervision 507
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 23 of 55
16737403-file00.docx
Writing - review & editing 508
509
Huanhuan Cui: 510
Investigation 511
Methodology 512
Writing - review & editing 513
514
Angela Luttick: 515
Investigation 516
Methodology 517
Writing - review & editing 518
519
Sean McDonald: 520
Conceptualisation 521
Formal analysis 522
Investigation 523
Methodology 524
Writing - review & editing 525
526
Arran Greenhalgh: 527
Conceptualisation 528
Formal analysis 529
Funding acquisition 530
Investigation 531
Methodology 532
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 24 of 55
16737403-file00.docx
Writing - review & editing 533
534
Jason Kwong: 535
Conceptualisation 536
Formal analysis 537
Funding acquisition 538
Methodology 539
Writing - original draft 540
Writing - review & editing 541
542
Norelle Sherry: 543
Conceptualisation 544
Formal analysis 545
Investigation 546
Methodology 547
Validation 548
Writing - original draft 549
Writing - review & editing 550
551
Maryza Graham: 552
Conceptualisation 553
Formal analysis 554
Investigation 555
Methodology 556
Validation 557
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 25 of 55
16737403-file00.docx
Writing - review & editing 558
559
Tuyet Hoang: 560
Conceptualisation 561
Funding acquisition 562
Investigation 563
Methodology 564
Validation 565
Writing - review & editing 566
567
Marion Herisse: 568
Conceptualisation 569
Funding acquisition 570
Investigation 571
Methodology 572
Resources 573
Validation 574
Writing - review & editing 575
576
Sacha Pidot: 577
Investigation 578
Methodology 579
Resources 580
Writing - review & editing 581
582
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 26 of 55
16737403-file00.docx
Deborah Williamson: 583
Conceptualisation 584
Formal analysis 585
Funding acquisition 586
Investigation 587
Methodology 588
Writing - review & editing 589
590
Benjamin Howden: 591
Conceptualisation 592
Formal analysis 593
Funding acquisition 594
Investigation 595
Methodology 596
Project administration 597
Resources 598
Writing - original draft 599
Writing - review & editing 600
601
Ian Monk: 602
Conceptualisation 603
Data curation 604
Formal analysis 605
Funding acquisition 606
Investigation 607
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 27 of 55
16737403-file00.docx
Methodology 608
Project administration 609
Resources 610
Validation 611
Writing - original draft 612
Writing - review & editing 613
614
Timothy Stinear: 615
Conceptualisation 616
Data curation 617
Formal analysis 618
Funding acquisition 619
Investigation 620
Methodology 621
Project administration 622
Resources 623
Software 624
Supervision 625
Validation 626
Visualisation 627
Writing - original draft 628
Writing - review & editing 629
630
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 28 of 55
16737403-file00.docx
8.2 Conflicts of interest 631
The authors declare the following conflict of interest: Nickala Best, Sean McDonald, and Arran 632
Greenhalgh are employees of GeneWorks, a commercial entity that distributes OptiGene reagents in 633
Australia. 634
635
8.3 Funding information 636
DAW is supported by an Emerging Leadership Investigator Grant from the National Health and 637
Medical Research Council (NHMRC) of Australia (APP1174555). TPS is supported by NHMRC 638
Research Fellowship (APP1105525). BPH is supported by NHMRC Practitioner Fellowship 639
(APP1105905). 640
641
8.4 Ethical approval 642
This study was conducted in accordance with the National Health and Medical Research Council of 643
Australia National Statement for Ethical Conduct in Human Research 2007 (Updated 2018). The 644
study was exempt from requiring specific approvals, as it involved the use of existing collections of 645
data or records that contained non-identifiable data about human beings [37]. 646
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 29 of 55
16737403-file00.docx
9. References 647
1. Victorian Department Health and Human Services 2020. Coronavirus disease 2019 648
(COVID-19) Guidelines for health services and general practitioners - Version 17, 649
5/04/2020. State Government of Victoria, Australia. 650
https://www.dhhs.vic.gov.au/health-services-and-general-practitioners-coronavirus-651
disease-covid-19 (accessed 7 April 2020) 652
2. UK National Health Service 2020. Guidance and standard operating procedure: COVID-19 653
virus testing in NHS laboratories, 16/03/2020. NHS, London. 654
https://www.england.nhs.uk/coronavirus/publication/guidance-and-standard-operating-655
procedure-covid-19-virus-testing-in-nhs-laboratories/ (accessed 7 April 2020) 656
3. Center for Disease Control. 2020. A 2019-Novel Coronavirus (2019-nCoV) real-time 657
RT-PCR panel primers and probes, 24/01/2020. US Department of Health & Human 658
Services, CDC, Atlanta. https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-659
pcr-panel-primer-probes.pdf (accessed 7 April 2020) 660
4. Centre for Disease Control. 2020. Real-time RT-PCR panel for detection 2019-Novel 661
Coronavirus, 24/01/2020. US Department of Health & Human Services, CDC, 662
Atlanta. https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-for-663
detection- instructions.pdf (accessed 7 April 2020). 664
5. Food and Drug Administration. 2020. Policy for diagnostic tests for Coronavirus 665
disease – 2019 during the public health emergency – Immediately in effect guidance 666
for clinical laboratories, commercial manufacturers, and food and Drug administration 667
staff, 16/03/2020. US Department of Health & Human Services, Food and Drug 668
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 30 of 55
16737403-file00.docx
Administration. https://www.fda.gov/regulatory-information/search-fda-guidance-669
documents/policy-diagnostic-tests-coronavirus-disease-2019-during-public-health-670
emergency (accessed 7 April 2020). 671
6. World Health Organization. 2020. Laboratory testing for coronavirus disease 2019 672
(COVID-19) in suspected human cases, 2/03/2020. World Health Organisation, 673
Geneva. https://apps.who.int/iris/bitstream/handle/10665/331329/WHO-COVID-19-674
laboratory-2020.4-eng.pdf?sequence=1&isAllowed=y (accessed 7 April 2020). 675
7. World Health Organization. 2020. Coronavirus disease (COVID-19) technical 676
guidance: Laboratory testing for 2019-nCoV in humans. 3. Molecular assays to 677
diagnose COVID-19, 03/2020. World Health Organisation, Geneva. 678
https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-679
guidance/laboratory-guidance (accessed 7 April 2020) 680
8. Kelly P. 2020. Deputy Chief Medical Officer’s press conference about COVID-19 on 16 681
March. Australian Government. https://www.health.gov.au/news/deputy-chief-medical-682
officers-press-conference-about-covid-19-on-16-march (accessed 29 April 2020). 683
9. Arnold C. COVID-19: Biomedical research in a world under social-distancing 684
measures. Nat Med 2020. doi:10.1038/d41591-020-00005-1. 685
10. Anonymous. Open for outbreaks. Nat Biotechnol 2020; 38:377. doi:10.1038/s41587-020-686
0499-y 687
11. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K et al. Loop-mediated 688
isothermal amplification of DNA. Nucleic Acids Res 2000; 28:E63. doi:10.1093/nar/28.12.e63 689
12. Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H et al. Development and evaluation of 690
a novel loop-mediated isothermal amplification method for rapid detection of severe acute 691
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 31 of 55
16737403-file00.docx
respiratory syndrome coronavirus. J Clin Microbiol 2004; 42:1956-61. doi: 692
10.1128/jcm.42.5.1956-1961.2004 693
13. Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct 694
detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum 695
samples. J Clin Microbiol 2003; 41:2616-22. doi: 10.1128/JCM.41.6.2616-2622.2003 696
14. Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification (LAMP): recent 697
progress in research and development. J Infect Chemother 2013; 19:404-11. doi: 698
10.1007/s10156-013-0590-0 699
15. Poon LL, Wong BW, Ma EH, Chan KH, Chow LM et al. Sensitive and inexpensive 700
molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from 701
heat-treated blood by loop-mediated isothermal amplification. Clin Chem 2006; 52:303-6. 702
doi: 10.1373/clinchem.2005.057901 703
16. Tsujimoto M, Nakabayashi K, Yoshidome K, Kaneko T, Iwase T et al. One-step nucleic acid 704
amplification for intraoperative detection of lymph node metastasis in breast cancer patients. 705
Clin Cancer Res 2007; 13:4807-16. doi: 10.1158/1078-0432.CCR-06-2512 706
17. Zhang Y, Odiwuor N, Xiong J, Sun L, Nyaruaba RO et al. Rapid Molecular Detection 707
of SARS-CoV-2 (COVID-19) Virus RNA Using Colorimetric LAMP. medRxiv 2020. 708
doi: 10.1101/2020.02.26.20028373. 709
18. Yu L, Wu S, Hao X, Li X, Liu X et al. Rapid colorimetric detection of COVID-19 710
coronavirus using a reverse tran-scriptional loop-mediated isothermal amplification 711
(RT-LAMP) diagnostic plat-form: iLACO. MedRxiv 2020. 712
doi:10.1101/2020.02.20.20025874. 713
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 32 of 55
16737403-file00.docx
19. Yan C, Cui J, Huang L, Du B, Chen L et al. Rapid and visual detection of 2019 novel 714
coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal 715
amplification assay. Clin Microbiol Infect 2020. doi:10.1016/j.cmi.2020.04.001. 716
20. Park G-S, Ku K, Baek S-H, Kim S-J, Kim S-I et al. Development of Reverse 717
Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays 718
Targeting SARS-CoV-2. J Mol Diagnostics 2020. doi: 10.1016/j.jmoldx.2020.03.006. 719
21. Pang J, Wang MX, Ang IYH, Tan SHX, Lewis RF et al.Potential Rapid Diagnostics, Vaccine 720
and Therapeutics for 2019 Novel Coronavirus (2019-nCoV): A Systematic Review. J Clin 721
Med 2020; 9:623. doi: 10.3390/jcm9030623 722
22. Osterdahl MF, Lee KA, Lochlainn ML, Wilson S, Douthwaite S et al. Detecting 723
SARS-CoV-2 at point of care: Preliminary data comparing Loop-mediated isothermal 724
amplification (LAMP) to PCR. MedRxiv. 2020. doi: 10.1101/2020.04.01.20047357. 725
23. Lu R, Wu X, Wan Z, Li Y, Zuo L et al. Development of a Novel Reverse Transcription Loop-726
Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2. Virol Sin 727
2020. doi: 10.1007/s12250-020-00218-1 728
24. Broughton JP, Deng X, Yu G, Fasching CL, Servellita V et al. CRISPR-Cas12-based 729
detection of SARS-CoV-2. Nat Biotechnol 2020. doi:10.1038/s41587-020-0513-4. 730
25. Caly L, Druce J, Roberts J, Bond K, Tran T et al. Isolation and rapid sharing of the 731
2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-732
19 in Australia. Med J Aust 2020. doi:10.5694/mja2.50569. 733
26. He H, Li R, Chen Y, Pan P, Tong W et al. Integrated DNA and RNA extraction using 734
magnetic beads from viral pathogens causing acute respiratory infections. Sci Rep 2017; 735
7:45199. doi: 10.1038/srep45199 736
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 33 of 55
16737403-file00.docx
27. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A et al. Detection of 2019 novel 737
coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25. doi: 10.2807/1560-738
7917.ES.2020.25.3.2000045 739
28. Baek YH, Um J, Antigua KJC, Park JH, Kim Y et al. Development of a reverse 740
transcription-loop-mediated isothermal amplification as a rapid early-detection 741
method for novel SARS-CoV-2. 2020. Emerg Microbes Infect 742
doi:10.1080/22221751.2020.1756698:1-31. 743
29. Nguyen T, Duong Bang D, Wolff A. 2019 Novel Coronavirus Disease (COVID-19): Paving 744
the Road for Rapid Detection and Point-of-Care Diagnostics. Micromachines (Basel) 2020; 745
11(3), 306. doi:10.3390/mi11030306. 746
30. FIND. 2020. SARS-CoV-2 diagnostic pipeline, 2020 https://www.finddx.org/covid-747
19/pipeline/ (accessed 29 April 2020). 748
31. Jayawardena S, Cheung CY, Barr I, Chan KH, Chen H et al. Loop-mediated isothermal 749
amplification for influenza A (H5N1) virus. Emerg Infect Dis 2007; 13:899-901. doi: 750
10.3201/eid1306.061572 751
32. Lopez-Jimena B, Wehner S, Harold G, Bakheit M, Frischmann S et al. Development of a 752
single-tube one-step RT-LAMP assay to detect the Chikungunya virus genome. PLoS Negl 753
Trop Dis 2018; 12:e0006448. doi: 10.1371/journal.pntd.0006448 754
33. Silva S, Paiva MHS, Guedes DRD, Krokovsky L, Melo FL et al. Development and Validation 755
of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid 756
Detection of ZIKV in Mosquito Samples from Brazil. Sci Rep 2019; 9:4494. doi: 757
10.1038/s41598-019-40960-5 758
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 34 of 55
16737403-file00.docx
34. Bhadra S, Riedel TE, Lakhotia S, Tran ND, Ellington AD. High-surety isothermal 759
amplification and detection of SARS-CoV-2, including with crude enzymes. BioRxiv 760
2020. doi: https://doi.org/10.1101/2020.04.13.039941. 761
35. Moore C, Corden S, Sinha J, Jones R. Dry cotton or flocked respiratory swabs as a simple 762
collection technique for the molecular detection of respiratory viruses using real-time 763
NASBA. J Virol Meth 2008; 153:84-9. doi: 10.1016/j.jviromet.2008.08.001 764
36. Kim EM, Jeon HS, Kim JJ, Shin YK, Lee YJ et al. Uracil-DNA glycosylase-treated reverse 765
transcription loop-mediated isothermal amplification for rapid detection of avian influenza 766
virus preventing carry-over contamination. J Vet Sci 2016; 17:421-5. doi: 767
10.4142/jvs.2016.17.3.421 768
37. National Health and Medical Research Council. 2018. National Statement on Ethical 769
Conduct in Human Research 2007 (Updated 2018). The National Health and Medical 770
Research Council, the Australian Research Council and Universities Australia. 771
Commonwealth of Australia, Canberra. https://nhmrc.gov.au/about-772
us/publications/national-statement-ethical-conduct-human-research-2007-updated-773
2018. 774
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 35 of 55
16737403-file00.docx
10. Figures and tables 775
776
Fig. 1. Limit of detection of N1 STOP-LAMP and establishing optimal sample template volume and 777
inactivation conditions. (A) Plot showing performance of the N1 LAMP assay across 5-log10 dilution 778
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 36 of 55
16737403-file00.docx
of purified viral RNA. Y-axis is time-to-positive (Tp) and x-axis is estimate of viral genomes/reaction 779
based on starting RNA concentration. The number of replicates per dilution (n) and number of 780
positive replicates per dilution is indicated. (B) Example N1-STOP-LAMP amplification plots for 781
SARS-CoV-2 positive clinical sample No. 52, showing inhibitory impact of 5 μL of a neat sample 782
matrix on LAMP and the effect of diluting the UTM in water. (C) N1-STOP-LAMP amplification 783
plots for three SARS-CoV-2 positive clinical samples, showing no loss in detection sensitivity after 784
specimen heat-treatment of 60°C for 30 min to inactivate virus. 785
786
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 37 of 55
16737403-file00.docx
787
Fig. 2: Comparison of E-gene RT-qPCR with N1-STOP-LAMP and limit of detection. Titred virus 788
was serially diluted 10-fold in UTM in triplicate and RNA was extracted from each replicate dilution. 789
A 1 µL aliquot of each UTM dilution was also tested directly by N1-STOP-LAMP. (A) Shows 790
calibration curve for the E-gene RT-qPCR. A curve was interpolated using linear regression. (B) 791
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 38 of 55
16737403-file00.docx
Comparative performance of N1-STOP-LAMP with 5 µL purified RNA versus 1 µL or 5 µL of UTM 792
directly added to the reaction. 793
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 39 of 55
16737403-file00.docx
794
Fig. 3: Clinical evaluation of N1-STOP-LAMP. (A) Establishing limit of detection according to FDA 795
Emergency Use Authorisations (EUA) guidelines. Twenty, 1 µL replicates of SARS-CoV-2 virus in 796
Universal Transport Medium (UTM) at a concentration of 54 TCID50 mL-1 were tested by direct N1-797
STOP-LAMP. All data points plotted, with average and 95% CI indicated. (B) Assessment of assay 798
against FDA EUA clinical performance criteria. Thirty healthy volunteer dry nasopharyngeal swabs 799
eluted in 1.5 mL of PBS were screened by N1-STOP-LAMP. Shown are time-to-positives (Tp). Mean 800
and 95% CI are indicated. (C). Plot showing correspondence between 107 E-gene RT-qPCR positive 801
and N1-STOP-LAMP, where 1 μL of clinical specimen in UTM was added directly to the RT-LAMP 802
reaction. Y-axis is LAMP Tp and x-axis is RT-qPCR cycle threshold (Ct). The dotted line indicates 803
Tp 30 mins. Results plotted above this line (encircled) were RT-qPCR positive but N1-STOP-LAMP 804
negative. (D) Results of 4-way interlaboratory comparison of N1-STOP-LAMP. Ten clinical samples 805
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 40 of 55
16737403-file00.docx
previously tested positive by E-gene RT-qPCR were used to create a test panel for comparing assay 806
performance between laboratories. 807
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 41 of 55
16737403-file00.docx
Table 1. FDA validation study recommendations for SARS-CoV-2 molecular diagnostics [5]. 808
Method Validation
Parameter Validation description and acceptance criteria
1. Limit of
Detection (LoD)
Test a dilution series of three replicates per concentration. LoD defined as lowest
concentration at which 19/20 replicates positive.
2. Clinical
evaluation
Test at least 30 contrived reactive specimens and 30 non-reactive specimens. At
least 20 specimens should have virus/RNA concentration of 1-2x LoD, remainder
spanning the assay range. Acceptance defined as 95% agreement 1-2x LoD, and
100% agreement at other concentrations and for negative specimens.
3. Inclusivity
In silico analysis indicating percent identity matches of target region against
publicly available SARS-CoV-2 sequences. Acceptance defined as 100% match
against all entries with the selected primers/probes.
4. Cross-reactivity
In silico analysis of the assay primers/probes compared to respiratory microbiota
and other viral pathogens. Acceptance defined as <80% homology between all
primers/probes and non-target sequences present in public databases.
809
Table 2. Assessment of heat treatment of universal transport medium specimens on detection 810
sensitivity. 811
Treatment
Sample No
E-gene RT-qPCR Ct
No heat
Tp (min:sec)
Heat
Tp (min:sec)
50 21.76 10:15 10:45
51 24.40 17:45 15:45
52 15.05 07:15 07:45
Ct = cycle threshold; Tp = time-to-positive 812
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 42 of 55
16737403-file00.docx
813
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 43 of 55
16737403-file00.docx
Table 3. Comparison of N1-STOP-LAMP and RT-qPCR performance using titred virus diluted in 814
universal transport medium. 815
N1-LAMP
(1 μL direct)
N1-LAMP
(5 μL direct)
N1-LAMP
(5 μL of 60 μL)
RT-qPCR
(5 μL of 60 μL)
SARS-CoV-2
(TCID50 mL-1) N
Tp
(Avg)
CV
(%)
Tp
(Avg)
CV
(%)
Tp
(Avg)
CV
(%)
Ct
(Avg)
CV
(%)
54000 3 06.33 02.28 06.41 02.25 05.33 02.71 18.41 0.33
5400 3 07.42 01.95 07.58 05.04 06.33 06.03 21.40 3.75
540 3 08.92 11.33 09.25 02.70 07.50 03.33 24.77 1.03
54 3 15.75 12.60 17.67 48.49 10.67 02.71 27.94 1.13
5.4 3 nd - 16.25 08.70# 16.88 07.33# 31.10 1.17
0.54 3 nd - nd nd nd - 36.96 2.79#
Tp = time-to-positive; CV = Coefficient of variation; nd = not detected; # 2/3 replicates positive at this dilution. 816
817
Table 4. Summary of clinical evaluation comparisons against FDA criteria. 818
LoD = Limit of detection; Tp = time-to-positive; # range indicated within parentheses. 819
820
Negative
1-2x LoD
Tp (min:sec)
2x LoD
Tp (min:sec)
N1-STOP-LAMP + 0 20 (07:30 – 22:00)# 10 (08:30 – 21:00)#
N1-STOP-LAMP - 30 0 0
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 44 of 55
16737403-file00.docx
Table 5. Comparison of N1-STOP-LAMP versus E-gene RT-qPCR using clinical specimens. 821
RT-qPCR + RT-qPCR - Total
N1-STOP-LAMP + 93 0 93
N1-STOP-LAMP - 14 50 64
Total 107 50 157
822
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 45 of 55
16737403-file00.docx
Supplementary data 823
Table S1. Clinical data positivity summary (107 E-gene RT-qPCR positives vs N1-STOP-LAMP Tp). 824
Sample
E-gene RT-qPCR
Ct
N1-STOP-LAMP
Tp (min:sec)
N1-STOP-LAMP retest of RNA
1 23.0 17:45
2 21.0 08:05
3 23.0 09:45
4 26.0 28:00
5 nd nd
6 21.2 11:00
7 17.8 10:00
8 16.3 08:15
9 19.3 08:45
10 18.6 08:30
11 25.1 20:00
12 22.0 13:45
13 28.1 14:00
14 23.2 11:00
15 24.3 20:15
16 28.3 29:15
17 23.1 17:30
18 26.1 28:15
19 16.4 07:45
20 20.9 11:45
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 46 of 55
16737403-file00.docx
21 27.2 16:45
22 20.1 10:15
23 20.4 11:00
24 20.0 08:45
25 18.9 09:00
26 21.6 11:45
27 27.1 27:00
28 21.9 16:00
29 17.0 09:45
30 15.4 09:00
31 19.3 17:15
32 20.6 11:15
33 15.1 09:30
34 27.6 20:30
35 28.5 21:45
36 16.3 09:15
37 16.5 09:45
38 23.1 22:30
39 31.5 nd
40 30.5 nd
41 25.9 23:15
42 21.3 13:30
43 30.6 29:15
44 25.4 15:45
45 22.8 13:15
46 27.2 29:15
47 21.7 08:30
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 47 of 55
16737403-file00.docx
48 16.5 07:15
49 20.4 08:30
50 21.8 10:15
51 24.4 12:00
52 15.1 08:00
53 28.8 27:00
54 18.2 07:45
55 17.2 06:00
56 26.7 09:00
57 23.9 11:30
58 16.2 06:30
59 17.8 07:15
60 27.8 24:15
61 27.2 11:45
62 35.8 nd
63 22.8 11:45
64 17.0 08:00
65 20.0 16:45
66 19.5 08:45
67 24.8 12:15
68 29.2 28:30
69 32.3 nd
70 26.6 20:30
71 33.1 28:00
72 20.0 07:30
73 18.6 08:30
74 17.5 07:50
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 48 of 55
16737403-file00.docx
75 30.7 25:30
76 20.9 13:00
77 19.0 07:15
78 26.3 11:45
79 22.6 08:30
80 29.3 nd
81 20.9 08:30
82 21.5 14:15
83 20.0 07:45
84 26.6 nd
85 28.5 nd
86 28.2 26:15
87 30.3 nd
88 27.6 13:15
89 20.0 09:15
90 27.4 27:45
91 18.7 07:15
92 30.0 20.30
93 28.0 nd 10:00
94 33.2 nd nd
95 19.7 08:45
96 28.2 nd 17:45
97 34.7 09:00
98 25.8 nd 25:30
99 21.3 12:15
100 16.5 07:45
101 33.4 25:15
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 49 of 55
16737403-file00.docx
102 25.1 19:00
103 20.3 09:45
104 26.8 nd 12:25
105 25.1 16:00
106 22.4 12:30
107 21.4 08:25
Ct = cycle threshold; Tp = time-to-positive 825
826
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 50 of 55
16737403-file00.docx
Supplementary Table S2. Results of N1-STOP-LAMP external quality assessment among four 827
laboratories. 828
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 51 of 55
16737403-file00.docx
Sample E-gene
RT-qPCR Ct
N1-STOP-LAMP Tp Agreement
with RT-qPCR Lab A Lab B Lab C Lab D
1 16.3 06.31 08:00 05:12 04:48 4/4
2 nd nd nd 19:54 nd 3/4
3 16.5 06.54 04:26 06:06 04:54 4/4
4 nd nd nd nd nd 4/4
5 20.4 08.51 08:44 08:12 07:24 4/4
6 nd nd nd nd nd 4/4
7 21.7 09.30 05:58 07:12 06:54 4/4
8 nd nd nd nd nd 4/4
9 22.1 12.3 12:06 11:36 07:00 4/4
10 nd nd nd nd nd 4/4
11 22.8 08.45 05:18 07:24 07:54 4/4
12 25.4 16.45 16:16 09:30 07:06 4/4
13 25.9 24.30 26:21 20:48 25:12 4/4
14 27.2 nd 24:32 nd nd 1/4
15 27.6 29.15 25:18 23:18 28:06 4/4
16 28.5 29.15 nd nd nd 1/4
17 29 nd nd nd nd 0/4
18 30.6 29.15 23:04 nd 24:00 3/4
19 nd nd nd nd nd 4/4
20 nd nd nd nd nd 4/4
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 52 of 55
16737403-file00.docx
Ct = cycle threshold; Tp = time-to-positive 829
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 53 of 55
16737403-file00.docx
Supplementary Table S3. N1-STOP-LAMP screen of NATtrol™ Respiratory Panel 2 (RP2) 830
Controls. 831
Virus/Organism Strain RP2
Control 1
RP2
Control 2
N1-STOP-
LAMP
Adenovirus Type 1 N/A Positive Negative Negative
Adenovirus Type 3 N/A Positive Negative Negative
Adenovirus Type 31 N/A Positive Negative Negative
C. pneumoniae CWL-029 Positive Negative Negative
Influenza A 2009 H1N1pdm A/NY/02/2009 Positive Negative Negative
Influenza A 2009 H3N2 A/Brisbane/10/07 Positive Negative Negative
Human Metapneumovirus Type 8 Peru6-2003 Positive Negative Negative
M. pneumoniae M129 Positive Negative Negative
Parainfluenza Type 1 N/A Positive Negative Negative
Parainfluenza Type 4 N/A Positive Negative Negative
Rhinovirus Type 1A N/A Positive Negative Negative
B. parapertussis A747 Negative Positive Negative
B. pertussis A639 Negative Positive Negative
Coronavirus 229E N/A Negative Positive Negative
Coronavirus HKU-1 Recombinant Negative Positive Negative
Coronavirus NL63 N/A Negative Positive Negative
Coronavirus OC43 N/A Negative Positive Negative
Influenza A H1N1 A/New Cal/20/99 Negative Positive Negative
Influenza B B/Florida/02/06 Negative Positive Negative
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 54 of 55
16737403-file00.docx
Parainfluenza Type 2 N/A Negative Positive Negative
Parainfluenza Type 3 N/A Negative Positive Negative
RSV Type A 2006 isolate Negative Positive Negative
832
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint
Page 55 of 55
16737403-file00.docx
Supplementary data file 1. Global initiative on sharing all influenza data (GISAID) submitters table. 833
.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted April 30, 2020. . https://doi.org/10.1101/2020.04.28.067363doi: bioRxiv preprint